Modified polymer particle, packing material and column for chromatography

ABSTRACT

An object of the present invention is to prevent an isolation of carboxyl group caused by hydrolysis of ester bond, which occurs when a polymer particle containing an ester bond is used in the presence of a strongly acidic or strongly alkaline solution to thereby improve the acid Ealkali durability of the polymer particle. When a polymer particle containing an ester bond is treated with an acid or alkali hydrous solution and thereby an ester bond which is located and exposed on the surface of the particle and is readily hydrolyzed is previously hydrolyzed to isolate a carboxyl group and then the free carboxyl group is capped by amidation, to thereby dissolve the above problem.

TECHNICAL FIELD

The present invention relates to a polymer particle improved in theacid.alkali durability, a modified packing material for chromatography,production and modification methods thereof and a chromatography columnusing the packing material.

BACKGROUND ART

Conventionally, acrylate-base and methacrylate-base polymer particleshave been used as an ion exchange resin or a packing material forchromatography of various types. However, these polymer particlescontain an ester bond in the molecule and use of such a polymer in anacid or alkali solution encounters decrease in the strength due tohydrolysis of the ester bond or use as a packing material forchromatography suffers from change of the analysis pattern or the likedue to the isolated carboxyl group Therefore, the pH range of a solutionwhich can be used is limited.

Use examples of the packing material for chromatography includechromatography for anion analysis. The anion chromatography includes asuppressor method (a method of using a suppressor) and a non-suppressormethod (a method of not using a suppressor). As the eluent, an alkalisolution at a pH of 10 or more, such as carbonate buffer or sodiumhydroxide, is used in the suppressor method, whereas a weakly acidicsolution such as p-hydroxybenzoic acid, phthalic acid and trimesic acidis used in the non-suppressor method. Therefore, the acrylate-base andmethacrylate-base packing materials are conventionally limited only touse as a packing material for non-suppressor method (see,JP-A-2000-221179 (the term “JP-A” as used herein means an “unexaminedpublished Japanese patent application”).

Also, a production method of anion exchanger, an anion exchanger and ameasuring method of anion using the exchanger, characterized in that anacrylate- or methacrylate-base ester bond-containing anion exchanger istreated with an alkali solution to decompose the ester bond and therebyproduce a hydroxyl group and/or a carboxyl group, are known (see,JP-A-2002-194117). However, in this case, the treatment of the esterbond-containing polymer with an acid or alkali hydrous solution isperformed for the purpose of isolating a hydroxyl group and/or acarboxyl group and adjusting the anion retention time at the analysis byusing the isolated group.

Other use examples of the packing material for chromatography includereverse phase chromatography. As the packing material for reverse phasecolumn, a silica-base particle such as octadecylated silica gel ispredominantly used. However, this packing material has a problem in thechemical stability and therefore, a polymer particle is also used.Examples of the acrylate-base or methacrylate-base packing materialinclude an ethylene glycol dimethacrylate/alkyl methacrylate copolymerand a glycerin dimethacrylate homopolymer having introduced thereinto anoctadecanoyl group.

In the silica-base particle, an unreacted silanol group remains in manycases and therefore, a separation performance sufficiently high as thereverse phase column may not be obtained. The degree in the residualamount of the silanol group can be examined by injecting an organic basesuch as pyridine in the packed column and comparing its peak shape. Itis generally known that as the number of residual silanol groups islarger, the peak is more tailed.

In order to solve the above-described problems encountered in use of asilica-base packing material, a method of capping the remaining silanolgroup with an inactive group is known. Recently, in order to perform thecapping at a higher density, for example, a method of reacting twoterminal addition agents each capable of partially forming a complexcompound in a closed container in an inert gas atmosphere(JP-A-10-72579) and a method of reacting the packing material with achemical modifier in a supercritical fluid (JP-A-9-49829) have beenreported.

On the other hand, the polymer particle-packed column is advantageous inthat the chemical stability is excellent as compared with the silicacolumn, however, this column is inferior to the silica column in that,for example, (1) the lot difference of retention volume is large, (2)the reproducibility of retention volume is bad, (3) the tailing isintensified, (4) the theoretical plate number is small. Therefore, thepolymer-base distribution-adsorption column is very small in the numberof products available on the market and is extremely limited in theopportunity where this column is used. Those problems seriously appearparticularly when an eluent completely free of a salt is used. Suchproblems of the polymer particle can be solved by a method of cappingthe free carboxyl group of a polymer-base packing material by aninactive group (see, JP-A-2000-310623). However, also in this case, theacid.alkali durability is not sufficiently high and the pH range of theeluent used is limited.

JP-A-2003-176363 describes a packing material obtained by covering apolymer particle with a crosslinkable epoxy compound and introducing acarbon chain through an ether bond, and this packing material isrevealed to have high acid.alkali durability. However, even in thiscase, when an acrylate-base or methacrylate-base particle is used as thebase material gel and a strongly acidic or strongly alkaline solution isused as the eluent, an ester bond exposed to the particle surface ishydrolyzed to produce a new free carboxyl group and therefore, theeluent pH range usable at the analysis is limited.

DISCLOSURE OF THE INVENTION

One object of the present invention is to provide a polymer particleimproved in the acid.alkali durability, which is used in performing aliquid chromatography analysis by using a polymer particle containing anester bond and which can be used even in a strongly acidic or stronglyalkaline solution. More specifically, one object of the presentinvention is to provide a polymer particle improved in the acid.alkalidurability, a modified packing material for chromatography, productionmethods thereof and a chromatography column using the packing material.

The present inventors have found that when a polymer particle containingan ester bond is treated with an acid or alkali hydrous solution andthereby an ester bond which readily comes into contact with liquid andis readily hydrolyzed is previously hydrolyzed to isolate a carboxylgroup and then the free carboxyl group is capped by amidation, theacid.alkali durability of the polymer particle is enhanced. The presentinvention has been accomplished based on this finding.

That is, the present invention relates, for example, to the followingmatters.

[1] A modified polymer particle improved in the acid.alkali durability,obtained by treating a polymer particle containing an ester bond with anacid or alkali hydrous solution to partially hydrolyze the ester bondand isolate a carboxyl group and capping the free carboxyl group throughamidation.

[2] The modified polymer particle improved in the acid.alkali durabilityas described in [1], wherein the ester bond having been hydrolyzed is anester bond readily coming into contact with liquid.

[3] The modified polymer particle improved in the acid-alkali durabilityas described in [1], wherein the amine used for the amidation is anamine represented by formula (1):NHR¹R²   (1)(wherein R¹ and R² each independently represents a hydrogen atom, analkyl group having a carbon number of 18 or less, which may be branchedor may be substituted by a halogen, or a phenyl group).

[4] The modified polymer particle improved in the acid.alkali durabilityas described in [3], wherein the amine used for the amidation is anamine represented by formula (2):NH₂R³   (2)(wherein R³ represents a hydrogen atom, an alkyl group having a carbonnumber of 18 or less, which may be branched or may be substituted by ahalogen, or a phenyl group).

[5] A modified polymer particle improved in the acid.alkali durability,wherein when the polymer particle is packed in a column having an innerdiameter of 4.6 mm and a length of 150 mm and the alkali durability isevaluated using the column by the following method, the increasepercentage of the pyridine retentivity after passing an alkali eluent is50% or less:

Evaluation Method:

(1) an eluent of acetonitrile/aqueous 0.1% phosphoric acidsolution=30/70 is passed at a flow rate of 0.3 ml/min for 30 minutes,the column temperature is set to 40° C., and the pyridine retentivity ismeasured by using an eluent of acetonitrile/water=30/70 at a flow rateof 0.5 ml/min,

(2) an alkali eluent of acetonitrile/aqueous 0.01 mol sodium hydroxidesolution=50/50 is passed through the same column at a flow rate of 0.5ml/min for 4 hours at a column temperature of 40° C., then an eluent ofacetonitrile/aqueous 0.1% phosphoric acid solution=30/70 is passed at aflow rate of 0.3 ml/min for 30 minutes, the column temperature is set to40° C., and the pyridine retentivity is measured by using an eluent ofacetonitrile/water=30/70 at a flow rate of 0.5 ml/min, and

(3) the pyridine retentivity of (2) is compared with the pyridineretentivity of (1).

[6] The modified polymer particle improved in the acid.alkali durabilityas described in any one of [1] to [5], wherein the average particle sizeof the polymer particle is from 1 to 50 μm.

[7] A polymer-base packing material for chromatography, using a modifiedpolymer particle improved in the acid.alkali durability, the polymerparticle being obtained by treating a polymer particle containing anester bond with an acid or alkali hydrous solution to partiallyhydrolyze the ester bond and isolate a carboxyl group and then cappingthe free carboxyl group through amidation.

[8] The polymer-base packing material for chromatography as described in[7], wherein the ester bond having been hydrolyzed is an ester bondreadily coming into contact with liquid.

[9] The polymer-base packing material for chromatography as described in[7], wherein the amine used for the amidation is an amine represented byformula (1):NHR¹R²   (1)(wherein R¹ and R² each independently represents a hydrogen atom, analkyl group having a carbon number of 18 or less, which may be branchedor may be substituted by a halogen, or a phenyl group).

[10] The polymer-base packing material for chromatography as describedin [9], wherein the amine used for the amidation is an amine representedby formula (2):NH₂R³   (2)(wherein R³ represents a hydrogen atom, an alkyl group having a carbonnumber of 18 or less, which may be branched or may be substituted by ahalogen, or a phenyl group).

[11] A polymer-base packing material for chromatography, using amodified polymer particle improved in the acid.alkali durability suchthat when the polymer particle is packed in a column having an innerdiameter of 4.6 mm and a length of 150 mm and the alkali durability isevaluated using the column by the following method, the increasepercentage of the pyridine retentivity after passing an alkali eluent is50% or less:

Evaluation Method:

(1) an eluent of acetonitrile/aqueous 0.1% phosphoric acidsolution=30/70 is passed at a flow rate of 0.3 ml/min for 30 minutes,the column temperature is set to 40° C., and the pyridine retentivity ismeasured by using an eluent of acetonitrile/water=30/70 at a flow rateof 0.5 ml/min,

(2) an alkali eluent of acetonitrile/aqueous 0.01 mol sodium hydroxidesolution=50/50 is passed through the same column at a flow rate of 0.5ml/min for 4 hours at a column temperature of 40° C., then an eluent ofacetonitrile/aqueous 0.1% phosphoric acid solution=30/70 is passed at aflow rate of 0.3 ml/min for 30 minutes, the column temperature is set to40° C., and the pyridine retentivity is measured by using an eluent ofacetonitrile/water=30/70 at a flow rate of 0.5 ml/min, and

(3) the pyridine retentivity of (2) is compared with the pyridineretentivity of (1).

[12] The polymer-base packing material for chromatography as describedin any one of [7] to [11], wherein the average particle size of thepolymer particle is from 1 to 50 μm.

[13] A method for producing a modified polymer particle improved in theacid.alkali durability, comprising treating a polymer particlecontaining an ester bond with an acid or alkali hydrous solution topartially hydrolyze the ester bond and isolate a carboxyl group and thencapping the free carboxyl group through amidation.

[14] The method for producing a modified polymer particle improved inthe acid.alkali durability as described in [13], wherein the ester bondhaving been hydrolyzed is an ester bond readily coming into contact withliquid.

[15] The method for producing a modified polymer particle improved inthe acid.alkali durability as described in [13], wherein the amine usedfor the amidation is an amine represented by formula (1):NHR¹R²   (1)(wherein R¹ and R² each independently represents a hydrogen atom, analkyl group having a carbon number of 18 or less, which may be branchedor may be substituted by a halogen, or a phenyl group).

[16] The method for producing a modified polymer particle improved inthe acid.alkali durability as described in [15], wherein the amine usedfor the amidation is an amine represented by formula (2):NH₂R³   (2)(wherein R³ represents a hydrogen atom, an alkyl group having a carbonnumber of 18 or less, which may be branched or may be substituted by ahalogen, or a phenyl group).

[17] The method for producing a modified polymer particle improved inthe acid.alkali durability as described in any one of [13] to [16],wherein when the polymer particle is packed in a column having an innerdiameter of 4.6 mm and a length of 150 mm and the alkali durability isevaluated using the column by the following method, the increasepercentage of the pyridine retentivity after passing an alkali eluent is50% or less:

Evaluation Method:

(1) an eluent of acetonitrile/aqueous 0.1% phosphoric acidsolution=30/70 is passed at a flow rate of 0.3 ml/min for 30 minutes,the column temperature is set to 40° C., and the pyridine retentivity ismeasured by using an eluent of acetonitrile/water=30/70 at a flow rateof 0.5 ml/min,

(2) an alkali eluent of acetonitrile/aqueous 0.01 mol sodium hydroxidesolution=50/50 is passed through the same column at a flow rate of 0.5ml/min for 4 hours at a column temperature of 40° C., then an eluent ofacetonitrile/aqueous 0.1% phosphoric acid solution=30/70 is passed at aflow rate of 0.3 ml/min for 30 minutes, the column temperature is set to40° C., and the pyridine retentivity is measured by using an eluent ofacetonitrile/water=30/70 at a flow rate of 0.5 ml/min, and

(3) the pyridine retentivity of (2) is compared with the pyridineretentivity of (1).

[18] The method for producing a modified polymer particle improved inthe acid.alkali durability as described in any one of [13] to [16],wherein the average particle size of the polymer particle is from 1 to50 μm.

[19] A method for producing a polymer-base packing material forchromatography, comprising producing the polymer-base packing materialfor chromatography described in any one of [6] to [12] by performing oneor both of the hydrolysis treatment with an acid or alkali hydroussolution and the capping treatment through amidation, in the state ofthe polymer-base packing material being packed in the column.

[20] A chromatography column using the polymer-base packing material forchromatography improved in the acid.alkali durability described in anyone of [6] to [12].

MODE FOR CARRYING OUT THE INVENTION

In the present invention, on performing liquid chromatography using apolymer particle containing an ester bond, an ester bond which readilycomes into contact with liquid and is readily hydrolyzed is previouslyhydrolyzed to isolate a carboxyl group and the carboxyl group is cappedthrough amidation, whereby the ester hydrolysis occurring during use inthe chromatography analysis is prevented, the acid.alkali durability ofthe polymer particle is improved, and a stable analysis can beperformed.

More specifically, in JP-A-2000-310623, a carboxyl group generated atthe polymerization step for production of a polymer particle is capped,whereas in the present invention, a readily hydrolyzable ester bond ispositively hydrolyzed and thereby the acid.alkali durability isremarkably improved.

The objective polymer particle to be modified by the method of thepresent invention is a polymer particle containing an ester bond in themolecule and examples thereof include a crosslinkable polymer containinga polyester structure. Furthermore, even in the case of a polymerparticle where the ester bond as a problem is contained in a part of thestructure, when the ester bond is considered to cause reduction of theacid.alkali durability, this is an objective polymer particle to bemodified by the method of the present invention.

Examples of the polymer particle containing a polyester structureinclude an ethylene glycol di(meth)acrylate/alkyl (meth)acrylatecopolymer, an ethylene glycol di(meth)acrylate homopolymer, a glycerindi(meth)acrylate/alkyl (meth)acrylate copolymer, a glycerindi(meth)acrylate homopolymer and a modified product thereof, an ethyleneglycol di(meth)acrylate/glycerin di(meth)acrylate copolymer and amodified product thereof, a trimethylol propane tri(meth)acrylate/alkyl(meta)acrylate copolymer, a trimethylol propane tri(meth)acrylatehomopolymer, a pentaerythritol tri(meth)acrylate/alkyl (meth)acrylatecopolymer and a modified product thereof, a pentaerythritoltri(meth)acrylate homopolymer and a modified product thereof, a vinyl(meth)acrylate homopolymer and a hydrolyzed or modified product of anoxirane ring of an ethylene glycol di(meth)acrylate/glycidyl(meth)acrylate copolymer.

Examples of the polymer particle where the ester bond as a problem iscontained in a part of the structure include those containing one or aplurality of partial structures selected from an amide, an imide, analcohol, an ether, an aromatic ring, an alkyl chain and the like at thesame time in addition to the above-described ester structures.

Such a polymer particle contains an ester bond and when the polymerparticle is used in an acid or alkali hydrous solution, the ester bondexposed to the particle surface is hydrolyzed. Therefore, this polymerparticle is low in the acid.alkali durability.

The modified polymer particle as referred to in the present inventionincludes all of the polymer particles containing an ester bondexemplified above, where the ester bond which is exposed to the surfaceand becomes readily hydrolyzable is previously cut by an acid or alkalihydrous solution and the produced free carboxyl group is capped throughamidation.

The amide bond is less hydrolyzable than the ester bond (see, PROTECTIVEGROUPS in ORGANIC SYNTHESIS, 3rd ed., page 442, middle column) andtherefore, when an ester bond readily coming into contact with liquid ispreviously cut and the produced free carboxyl group is capped throughamidation and converted into an amide bond, the production of a carboxylgroup can be prevented even if the polymer particle is used in an acidor alkali solution, and the durability of the polymer particle isenhanced.

That is, the ester bond readily coming into contact with liquid asreferred to in the present invention is an ester bond present relativelynear the polymer surface. By converting such an ester bond into an amidebond, the polymer surface is protected by a less hydrolyzable amide bondas described above, as a result, the ester bond not hydrolyzed by anacid or alkali treatment becomes more difficult of coming into contactwith liquid on use as a column or the like and thereby, the polymerparticle is considered to increase in the acid.alkali durability.

In the present invention, the polymer particle as an objective of themodification may have various sizes according to use. In the case of useas a packing material for chromatography, the polymer particlepreferably has an average particle size of 1 to 50 μm, more preferablyfrom 1.5 to 30 μm, still more preferably from 2 to 10 μm. If the averageparticle size is less than 1 μm, the column pressure excessivelyincreases to surpass the strength limit of the particle and this is notpreferred, whereas if the average particle size exceeds 50 μm, theseparation ability is low and this is not preferred in practice.

The method for the treatment with an acid or alkali hydrous solutionperformed in the present invention so as to cut the readily hydrolyzableester bond which is considered to be exposed to the polymer particlesurface is specifically described below.

As the acid hydrous solution, an aqueous solution of hydrochloric acid,sulfuric acid or the like can be used. In the case where the polymerparticle is less wettable to such an aqueous solution, a water-solubleorganic solvent of not undergoing a reaction in a strongly acidicsolution, such as dimethylsulfoxide, may be added in an appropriateamount before use of the aqueous solution. The concentration is notparticularly limited as long as the ester bond on the polymer particlesurface can be hydrolyzed, but the polymer particle is suspended byappropriately controlling the concentration such that the solution has apH of 3 or less, preferably from 1 to 2. The reaction temperature is notparticularly limited as long as the ester bond on the polymer particlesurface can be hydrolyzed, but the reaction temperature is suitably setto 20 to 100° C., preferably from 30 to 80° C., more preferably from 40to 60° C. The reaction term is also not particularly limited as long asthe ester bond on the polymer particle surface can be hydrolyzed, butthe reaction is suitably performed with stirring for 30 minutes to 24hours, preferably from 30 minutes to 10 hours, more preferably from 1 to6 hours.

As the alkali hydrous solution, an aqueous solution of a hydroxide suchas sodium hydroxide and potassium hydroxide can be used. In the casewhere the polymer particle is less wettable to such an aqueous solution,a water-soluble organic solvent of not undergoing a reaction in astrongly alkaline solution, such as dimethylsulfoxide, may be added inan appropriate amount before use of the aqueous solution. Theconcentration is not particularly limited as long as the ester bond onthe polymer particle surface can be hydrolyzed, but the polymer particleis suspended by appropriately controlling the concentration such thatthe solution has a pH of 10 to 14, preferably from 11 to 13. Thereaction temperature is not particularly limited as long as the esterbond on the polymer particle surface can be hydrolyzed, but the reactiontemperature is suitably set to 20 to 100° C., preferably from 30 to 80°C., more preferably from 40 to 60° C. The reaction term is also notparticularly limited as long as the ester bond on the polymer particlesurface can be hydrolyzed, but the reaction is suitably performed withstirring for 20 minutes to 24 hours, preferably from 30 minutes to 10hours, more preferably from 1 to 6 hours.

These ranges of pH, reaction temperature and reaction term in theconditions of hydrolysis of the ester bond may be individually adjustedand used, but all conditions are preferably adjusted in combination.

The method for the amidation used to achieve capping in the presentinvention is specifically described below.

The amine used for the amidation is basically not limited as long as thefree carboxyl group can be capped, but on considering the reactivity atthe amidation, the limitation of introduction due to steric hindrance,and the stability after the conversion into an amide, the amine usedtherefor is preferably an amine represented by formula (1):NHR¹R²   (1)(wherein R¹ and R² each independently represents a hydrogen atom, analkyl group having a carbon number of 18 or less, which may be branchedor may be substituted by a halogen, or a phenyl group).

The amine is more preferably a primary amine represented by formula (2):NH₂R³   (2)(wherein R³ represents a hydrogen atom, an alkyl group having a carbonnumber of 18 or less, which may be branched or may be substituted by ahalogen, or a phenyl group).

A smaller substituent size is advantageous in that the introductionefficiency increases due to small steric hindrance and thehydrophobicity of the polymer particle before modification is notlargely changed. However, in the case where the amount of the freecarboxyl group to be capped is very small, the hydrophobicity is lesschanged even when a relatively large inactive group is introduced,therefore, a substituent having a large size to a certain extent can beused. More specifically, in the case of an alkylamine, the alkyl grouppreferably has a carbon number of 18 or less, more preferably 12 orless, still more preferably 6 or less, and particularly preferably 4 orless.

Examples thereof include ammonia, methylamine, ethylamine,1-propylamine, isopropylamine, 1-butylamine, isobutylamine,tert-butylamine, 1-hexylamine, cyclohexyl-amine, 1-octylamine,dimetylamine, dietylamine and di(1-propyl) amine.

For the amidation in the present invention, a method commonly used inthe peptide synthesis or the like can be employed. However, it isnecessary that the portion except for the free carboxyl group of apolymer particle to be modified is not adversely affected. Furthermore,from the practical aspects such as introduction efficiency, ease ofhandling and cost, the amidation is preferably performed by using thefollowing combination of reagents.

(A) (i) A reagent for the production of a mixed acid anhydride, such asethyl chloroformate; a tertiary amine such as triethylamine; and anorganic solvent such as chloroform, and then (ii) an amine representedby formula (1).

(B) An amine represented by formula (2) such as 1-propylamine; adehydration condensing agent such as N,N′-diisopropylcarbodiimide; andan organic solvent such as toluene and N,N-dimethylformamide.

The modification method of a polymer particle containing an ester bondof the present invention is characterized in that a polymer particle istreated with the above-described acid or alkali hydrous solution andfurther brought into contact with a solution or suspension containingthose amidation reagents.

That is, a readily hydrolyzable ester bond which is considered to beexposed to the polymer particle surface is first hydrolyzed by treatingthe polymer particle with an acid or alkali hydrous solution and thenthe isolated carboxyl group is converted into an amide group bycontacting the polymer particle with a solution or suspension containingamidation reagents.

The polymer particle subjected to modification may be or may not bepreviously packed in a column. More specifically, one or both of thehydrolysis treatment by the acid or alkali hydrous solution and thecapping treatment through amidation may be performed in the state of thepolymer particle being packed in a column. For treating the polymerparticle in the packed state, a method of feeding a solution orsuspension containing the reagents to the column is used. The patternregarding the feeding amount, temperature, rate and time, the standingconditions after feeding, and the like are appropriately selected asneeded to complete the reaction.

With a conventional liquid chromatography column packed with a polymerparticle containing an ester bond readily coming into contact withliquid, the elution time and peak shape of an organic base such aspyridine sometimes greatly change between before and after the passingof a strongly acidic or strongly alkaline eluent. On the other hand,with a liquid chromatography column packed with the polymer particle ofthe present invention, the elution time and peak shape of an organicbase such as pyridine are remarkably suppressed from changing betweenbefore and after the passing of a strongly acidic or strongly alkalineeluent.

The problems encountered in the measurement using a conventional liquidchromatography column packed with a polymer particle containing an esterbond readily coming into contact with liquid is considered ascribable tothe production of a free carboxyl group resulting from the hydrolysis ofan ester bond exposed to the polymer particle surface at the passing ofa strongly acidic or strongly alkaline eluent. As the free carboxylgroup, a proton-type (R—COOH) carboxyl group and a metal-type (R—COOM)carboxyl group are considered to exist together. An organic base such aspyridine (the organic base is hereinafter referred to as “pyridine”) isgreatly adsorbed mainly to the former. This is because the pH is locallylow in the vicinity of the proton-type carboxylic acid (R—COOH) and theequilibrium of pyridine and pyridinium ion inclines to the pyridiniumion, as a result, an ionic bond of R—COO(−)→HNC₅H₅(+) is readilygenerated. If an eluent completely free of a salt is used, thisphenomenon more noticeably appears, because if a slight amount of metalis mixed in an eluent, the metal is captured on passing near theproton-type carboxylic acid (R—COOH) and concentrated in the column, asa result, a metal-type carboxylic acid (R—COOM) increases and pyridineis less adsorbed. In order to accurately evaluate the alkali durability,the metal-type carboxylic acid (R—COOM) need be converted into aproton-type carboxylic acid (R—COOH). For this purpose, an acid solutioncontaining phosphoric acid may be passed through the column beforeanalyzing pyridine.

From these points, when the pyridine analysis is performed before andafter passing a strongly acidic or strongly alkaline eluent through theobjective column to be evaluated, the effect of modification can beclearly detected.

More specifically, when an ester bond is hydrolyzed and a free carboxylgroup is produced, the retention volume of pyridine increases and thepeak shape tails, therefore, the effect of the present invention can bedetected by comparing the polymer particle after modification accordingto the present invention with the polymer particle before modification.

By this method, the effect on the acid.alkali durability of a polymerparticle, which is attained by the modification comprising a hydrolysistreatment with an acid or alkali hydrous solution and a cappingtreatment through amidation, can be verified.

EXAMPLES

The present invention is described in greater detail below by referringto Examples, however, the present invention is not limited to theseExamples. In Example 1, the production process of a polymer-base packingmaterial suitable for reverse phase liquid chromatography column isdescribed all the way from the initial preparation of base material gelto the final introduction of carbon chain, but steps 3 and 4 are theportions substantially concerned in the present invention. As such, themethod of the present invention can be utilized by inserting it on theway in a series of steps for producing a polymer particle.

Example 1

<Step 1: Synthesis of Base Material Crosslinked Polymer Particle>

In a mixed solution containing 2,000 g of glycerin dimethacrylate and900 g of 1-hexanol, 30 g of 2,2′-azobis(isobutyronitrile) was dissolvedto prepare an oil phase. Separately, 180 g of polyvinyl alcohol (KURARAYPOVAL PVA-224, produced by Kuraray Co., Ltd.) was dissolved in 3 literof water and thereto, 7 liter of water and subsequently a solutionobtained by dissolving 240 g of sodium chloride in 2 liter of water anda solution obtained by dissolving 15 g of sodium dodecylsulfate in 485ml of water were added and mixed to prepare an aqueous phase. Theobtained oil phase and aqueous phase were mixed in a 20 L-volumestainless steel-made container and dispersed in a high-speed disperser(homogenizer) by adjusting the rotation number and dispersion time suchthat the maximum particle size of oil droplet became 3 μm.

The obtained dispersion was reacted at 70° C. for 4 hours while stirringat 150 rpm. The crosslinked polymer particle produced was centrifuged(at 2,000 rpm for 10 minutes) and after discarding the supernatant, theprecipitate was dispersed (by using an ultrasonic washer) in 12 liter ofwarm water at 70° C. and then stirred at 70° C. for 3 hours. Theresulting solution was filtered by suction and the gel on the funnel waswashed with 60 liter of warm water at 70° C. and then with 18 liter ofacetone, air-dried by spreading it in a stainless steel-made vat, andfurther dried under reduced pressure at 60° C. for 24 hours. The polymerparticles obtained were classified by a pneumatic classifier to obtain716 g of crosslinked polymer particles having a weight average particlesize of 3 μm (hereinafter, this polymer particle is referred to as a“base material gel”).

<Step 2: Thorough Washing>

Pure water (500 ml) was added to 50 g of the base material gel obtainedin the step 1 and stirred under heating at 60° C. for 5 hours.Thereafter, the particles were collected by filtration, washed insequence with 2,000 ml of warm water at 70° C. and then with 300 ml ofmethanol, then air-dried by spreading the particles in a stainlesssteel-made vat, and further dried under reduced pressure at 60° C. for24 hours to obtain 49 g of a thoroughly washed base material gel.

<Step 3: Hydrolysis Treatment with Alkali Hydrous Solution>

The thoroughly washed base material gel (10 g) obtained in the step 2was dispersed in 50 ml of an aqueous 0.1N sodium hydroxide solution andstirred at 50° C. for 2 hours. The reaction liquid was filtrated bysuction and the gel on the funnel was washed in sequence with 100 ml of0.01N hydrochloric acid solution, with 500 ml of water and with 100 mlof acetone, then air-dried by spreading the gel in a stainlesssteel-made vat and further dried under reduced pressure at 60° C. for 3hours to obtain 10 g of a hydrolyzed gel.

<Step 4: Capping through Amidation>

The hydrolyzed gel (10 g) obtained in the step 3 was dispersed in 50 mlof chloroform at 25° C. While stirring the dispersion in a water bath at25° C., 2.3 g of triethylamine was added, after 5 minutes, 2.5 g ofethyl chloroformate was added, and after 30 minutes therefrom, 1.6 g of1-propylamine was added. Then, these were reacted for 3 hours. Thereaction mixture was filtrated by suction and the gel on the funnel waswashed in sequence with 100 ml of chloroform, with 500 ml of water andwith 100 ml of acetone, then air-dried by spreading the gel in astainless steel-made vat, and further dried under reduced pressure at60° C. for 3 hours to obtain 10 g of an amidated and capped gel.

<Step 5: Surface Crosslinking and Epoxy Ring-Opening Reaction>

The amidated and capped gel (10 g) obtained in the step 4 and 1 g ofethylene glycol diglycidyl ether were added to 50 ml of toluene andwhile stirring these in a water bath at 40° C., 0.5 g of borontrifluoride diethyl ether complex was added and reacted for 3 hours. Thereaction mixture was filtrated by suction and the gel on the funnel waswashed with 100 ml of acetone and then with 500 ml of water. The gel wastransferred to a reaction vessel and thereto, 50 ml of 0.1N hydrochloricacid was added and reacted at 50° C. for 1 hour. The reaction mixturewas filtered by suction and the gel on the funnel was washed with 500 mlof water and then with 100 ml of acetone, thereafter air-dried byspreading the gel in a stainless steel-made vat, and further dried underreduced pressure at 60° C. for 3 hours to obtain 11.5 g of a surfacecrosslinked gel.

<Step 6: Reaction for C18 Formation>

The surface crosslinked gel (10 g) obtained in the step 5 and 3 g ofoctadecyl glycidyl ether were dispersed in 100 ml of toluene and whilestirring in a water bath at 40° C., 0.5 g of boron trifluoride diethylether complex was added and reacted for 3 hours. The reaction mixturewas filtrated by suction and the gel on the funnel was washed insequence with 100 ml of toluene, with 300 ml of tetra-hydrofuran, with500 ml of water and with 100 ml of acetone, then air-dried by spreadingthe gel in a stainless steel-made vat, and further dried under reducedpressure at 60° C. for 3 hours to obtain 10.5 g of a carbonchain-introduced gel.

<Packing of Modified Gel>

The carbon chain-introduced gel obtained in the step 6 was packed in astainless steel-made column of 4.6 mm (inner diameter)×150 mm (length)by a slurry method to produce a reverse phase chromatography column(hereinafter called “Column A”).

Comparative Example 1

A column was produced by excluding the steps 3 and 4 in the columnproduction process of Example 1 (hereinafter called “Column B”).

Comparative Example 2

A column was produced by excluding the step 4 in the column productionprocess of Example 1 (hereinafter called “Column C”).

(1. Comparison of Alkali Durability)

Example 1 (Column A) and Comparative Example 1 (Column B) each wasevaluated on the alkali durability by the following method. For clearlyshowing the effect by the capping through amidation, the pyridine/phenoltest results of Comparative Example 2 (Column C) are also shown.

1-1. Alkali Durability Evaluation Method

1-1-1. Pyridine/Phenol Test in Initial State

An acidic eluent was passed through the test objective column under thefollowing conditions. This was performed so as to partialize a freecarboxyl group, if present, to a proton type (RCOOH).

-   Eluent: CH₃CN/aqueous 0.1% phosphoric acid solution=30/70 (v/v)-   Flow rate: 0.3 ml/min-   Column temperature: 25° C.-   Eluent passing time: 30 minutes

Subsequently, the conditions were changed to the following conditionsand after the liquid displacement for 30 minutes or more, pyridine andphenol were analyzed.

-   Eluent: CH₃CN/H₂O=30/70 (v/v)-   Flow rate: 1.00 ml/min-   Column temperature: 40° C.    1-1-2. Passing of Strongly Alkaline Eluent

Through the test objective column after the completion of apyridine/phenol test in the initial state, a strongly alkaline eluentwas passed under the following conditions. By this operation, an esterbond readily coming into contact with liquid was, if present, hydrolyzedto produce a free carboxyl group.

-   Eluent: CH₃CN/0.01N—NaOH (pH: 12)=50/50 (v/v)-   Flow rate: 0.50 ml/min-   Column temperature: 40° C.-   Eluent passing time: 4 hours    1-1-3. Pyridine/Phenol Test after Passing of Strongly Alkaline    Eluent

Using the test objective column after the passing of a strongly alkalineeluent, a pyridine/phenol test was performed by the same procedure as inabove 1-1-1.

1-2. Alkali Durability Evaluation Results

The evaluation results of Column A and Column B are shown in Table 1. Inthe Table, the retentivity k′ was calculated from a non-retention timet₀ and a compound retention time t_(r) according to the followingformula:k′=(t _(r)-t ₀)/t ₀

The tailing coefficient T was calculated from a peak width W_(0.05h) ata peak height of 5% and a peak width f in the peak rising side accordingto the following formula:T=W _(0.05h) /f

TABLE 1 Pyridine Phenol Pyridine Tailing Retentivity k′ Retentivity k′Coefficient After After After Passing of Passing of Passing of StronglyStrongly Strongly Initial Alkaline Initial Alkaline Initial AlkalineState Eluent State Eluent State Eluent Column A 1.5 1.6 3.6 3.6 0.750.75 Column B 1.6 2.5 3.6 3.6 1.5 4.3 Column C 4.7 — 3.6 — 5.8 —

In both of Column A and Column B, the phenol retentivity k′ was notchanged between the initial state and after passing of strongly alkalineeluent, but in Column B, the pyridine retentivity was increased by 56%and the tailing coefficient was also increased. This seems to revealthat an ester bond was hydrolyzed during the passing of a stronglyalkaline eluent and a free carboxyl group was produced. On the contrary,in Column A, the pyridine peak was scarcely changed and this seems toreveal that the effect of the present invention was noticeably broughtout.

Furthermore, by comparing the pyridine retentivity and the tailingcoefficient in the initial state of Column A with those of Column C, itcan be confirmed that the carboxyl group produced by the alkalitreatment in the production process of a packing material was amidatedand thereby capped.

(2. Comparison of Acid Durability)

Example 1 (Column A) and Comparative Example 1 (Column B) were evaluatedon the acid durability by the following method.

2-1. Acid Durability Evaluation Method

2-1-1. Pyridine/Phenol Test in Initial State

The pyridine/phenol test was performed by the same procedure as in above1-1-1.

2-1-2. Passing of Strongly Acidic Eluent

Through the test objective column after the completion of apyridine/phenol test in the initial state, a strongly acidic eluent waspassed under the following conditions. By this operation, an ester bondreadily coming into contact with liquid was, if present, hydrolyzed toproduce a free carboxyl group.

-   Eluent: MeOH/aqueous 1% trifluoroacetic acid solution (pH:    1.2)=10/90 (v/v)-   Flow rate: 1.00 ml/min-   Column temperature: 40° C.-   Eluent passing time: 70 hours    2-1-3. Pyridine/Phenol Test after Passing of Strongly Acidic Eluent

Using the test objective column after the passing of a strongly acidiceluent, a pyridine/phenol test was performed by the same procedure as inabove 1-1-1.

2-2. Acid Durability Evaluation Results

The evaluation results of Column A and Column B are shown in Table 2. Inthe Table, the retentivity k′ was calculated from a non-retention timet₀ and a compound retention time t_(r) according to the followingformula:k′=(t _(r)-t ₀)/t ₀

The tailing coefficient T was calculated from a peak width W_(0.05h) ata peak height of 5% and a peak width f in the peak rising side accordingto the following formula:T=W _(0.05h) /f

TABLE 2 Pyridine Phenol Pyridine Tailing Retentivity k′ Retentivity k′Coefficient After After After Passing of Passing of Passing of StronglyStrongly Strongly Initial Acidic Initial Acidic Initial Acidic StateEluent State Eluent State Eluent Column A 1.4 1.6 3.6 3.6 0.75 0.75Column B 1.6 2.6 3.6 3.6 1.5 3.6

In both of Column A and Column B, the phenol retentivity was not changedbetween-the initial state and after passing of strongly acidic eluent,but in Column B, the pyridine retentivity was increased by 63% and thetailing coefficient was also increased. This seems to reveal that anester bond was hydrolyzed during the passing of a strongly acidic eluentand a free carboxyl group was produced. On the contrary, in Column A,the pyridine peak was scarcely changed and this seems to reveal that theeffect of the present invention was noticeably brought out.

The polymer particle modified by the method of the present invention waspacked to produce a column for chromatography and organic bases such aspyridine were analyzed before and after the passing of a strongly acidicor strongly alkaline eluent and compared, as a result, the increase inretentivity and the tailing of peak were noticeably suppressed and theeffect of the present invention could be confirmed.

INDUSTRIAL APPLICABILITY

In the modified polymer particle of the present invention, an ester bondpresent near the surface and readily coming into contact with liquid ishydrolyzed and a carboxyl group produced is converted into an amidegroup, therefore, after the treatment, a less hydrolyzable amide bond ispresent on the surface and the remaining ester bond becomes difficult tocontact with liquid, as a result, the acid.alkali durability isconsidered to increase.

The polymer particle of the present invention can be used as a packingmaterial for chromatography and a column packed with this polymerparticle exhibits high acid.alkali durability and therefore, is usefulover a wide range of fields, such as separation and analysis of medicalor agrochemical preparations, food additives, intermediates thereof,natural or synthetic polymers, additives thereof, and environmentalpollutants.

1. A modified polymer particle improved in the acid.alkali durability,obtained by treating a polymer particle containing an ester bond with anacid or alkali hydrous solution to partially hydrolyze the ester bondand isolate a carboxyl group and capping the free carboxyl group throughamidation.
 2. The modified polymer particle improved in the acid.alkalidurability as claimed in claim 1, wherein the ester bond having beenhydrolyzed is an ester bond readily coming into contact with liquid. 3.The modified polymer particle improved in the acid.alkali durability asclaimed in claim 1, wherein the amine used for the amidation is an aminerepresented by formula (1):NHR¹R²   (1) (wherein R¹ and R² each independently represents a hydrogenatom, an alkyl group having a carbon number of 18 or less, which may bebranched or may be substituted by a halogen, or a phenyl group).
 4. Themodified polymer particle improved in the acid.alkali durability asclaimed in claim 3, wherein the amine used for the amidation is an aminerepresented by formula (2):NH₂R³   (2) (wherein R³ represents a hydrogen atom, an alkyl grouphaving a carbon number of 18 or less, which may be branched or may besubstituted by a halogen, or a phenyl group).
 5. A modified polymerparticle improved in the acid.alkali durability, wherein when thepolymer particle is packed in a column having an inner diameter of 4.6mm and a length of 150 mm and the alkali durability is evaluated usingthe column by the following method, the increase percentage of thepyridine retentivity after passing an alkali eluent is 50% or less:Evaluation Method: (1) an eluent of acetonitrile/aqueous 0.1% phosphoricacid solution=30/70 is passed at a flow rate of 0.3 ml/min for 30minutes, the column temperature is set to 40° C., and the pyridineretentivity is measured by using an eluent of acetonitrile/water=30/70at a flow rate of 0.5 ml/min, (2) an alkali eluent ofacetonitrile/aqueous 0.01 mol sodium hydroxide solution=50/50 is passedthrough the same column at a flow rate of 0.5 ml/min for 4 hours at acolumn temperature of 40° C., then an eluent of acetonitrile/aqueous0.1% phosphoric acid solution=30/70 is passed at a flow rate of 0.3ml/min for 30 minutes, the column temperature is set to 40° C., and thepyridine retentivity is measured by using an eluent ofacetonitrile/water=30/70 at a flow rate of 0.5 ml/min, and (3) thepyridine retentivity of (2) is compared with the pyridine retentivity of(1).
 6. The modified polymer particle improved in the acid-alkalidurability as claimed in any one of claims 1 to 5, wherein the averageparticle size of the polymer particle is from 1 to 50 μm.
 7. Apolymer-base packing material for chromatography, using a modifiedpolymer particle improved in the acid.alkali durability, the polymerparticle being obtained by treating a polymer particle containing anester bond with an acid or alkali hydrous solution to partiallyhydrolyze the ester bond and isolate a carboxyl group and then cappingthe free carboxyl group through amidation.
 8. The polymer-base packingmaterial for chromatography as claimed in claim 7, wherein the esterbond having been hydrolyzed is an ester bond readily coming into contactwith liquid.
 9. The polymer-base packing material for chromatography asclaimed in claim 7, wherein the amine used for the amidation is an aminerepresented by formula (1):NHR¹R²   (1) (wherein R¹ and R each independently represents a hydrogenatom, an alkyl group having a carbon number of 18 or less, which may bebranched or may be substituted by a halogen, or a phenyl group).
 10. Thepolymer-base packing material for chromatography as claimed in claim 9,wherein the amine used for the amidation is an amine represented byformula (2):NH₂R³   (2) (wherein R³ represents a hydrogen atom, an alkyl grouphaving a carbon number of 18 or less, which may be branched or may besubstituted by a halogen, or a phenyl group).
 11. A polymer-base packingmaterial for chromatography, using a modified polymer particle improvedin the acid-alkali durability such that when the polymer particle ispacked in a column having an inner diameter of 4.6 mm and a length of150 mm and the alkali durability is evaluated using the column by thefollowing method, the increase percentage of the pyridine retentivityafter passing an alkali eluent is 50% or less: Evaluation Method: (1) aneluent of acetonitrile/aqueous 0.1% phosphoric acid solution=30/70 ispassed at a flow rate of 0.3 ml/min for 30 minutes, the columntemperature is set to 40° C., and the pyridine retentivity is measuredby using an eluent of acetonitrile/water=30/70 at a flow rate of 0.5ml/min, (2) an alkali eluent of acetonitrile/aqueous 0.01 mol sodiumhydroxide solution=50/50 is passed through the same column at a flowrate of 0.5 ml/min for 4 hours at a column temperature of 40° C., thenan eluent of acetonitrile/aqueous 0.1 % phosphoric acid solution=30/70is passed at a flow rate of 0.3 ml/min for 30 minutes, the columntemperature is set to 40° C., and the pyridine retentivity is measuredby using an eluent of acetonitrile/water=30/70 at a flow rate of 0.5ml/min, and (3) the pyridine retentivity of (2) is compared with thepyridine retentivity of (1).
 12. The polymer-base packing material forchromatography as claimed in any one of claims 7 to 11, wherein theaverage particle size of the polymer particle is from 1 to 50 μm.
 13. Amethod for producing a modified polymer particle improved in theacid.alkali durability, comprising treating a polymer particlecontaining an ester bond with an acid or alkali hydrous solution topartially hydrolyze the ester bond and isolate a carboxyl group and thencapping the free carboxyl group through amidation.
 14. The method forproducing a modified polymer particle improved in the acid.alkalidurability as claimed in claim 13, wherein the ester bond having beenhydrolyzed is an ester bond readily coming into contact with liquid. 15.The method for producing a modified polymer particle improved in theacid-alkali durability as claimed in claim 13, wherein the amine usedfor the amidation is an amine represented by formula (1):NHR¹R²   (1) (wherein R¹ and R² each independently represents a hydrogenatom, an alkyl group having a carbon number of 18 or less, which may bebranched or may be substituted by a halogen, or a phenyl group).
 16. Themethod for producing a modified polymer particle improved in theacid.alkali durability as claimed in claim 15, wherein the amine usedfor the amidation is an amine represented by formula (2):NH₂R³   (2) (wherein R³ represents a hydrogen atom, an alkyl grouphaving a carbon number of 18 or less, which may be branched or may besubstituted by a halogen, or a phenyl group).
 17. The method forproducing a modified polymer particle improved in the acid-alkalidurability as claimed in any one of claims 13 to 16, wherein when thepolymer particle is packed in a column having an inner diameter of 4.6mm and a length of 150 mm and the alkali durability is evaluated usingthe column by the following method, the increase percentage of thepyridine retentivity after passing an alkali eluent is 50% or less:Evaluation Method: (1) an eluent of acetonitrile/aqueous 0.1% phosphoricacid solution=30/70 is passed at a flow rate of 0.3 ml/min for 30minutes, the column temperature is set to 40° C., and the pyridineretentivity is measured by using an eluent of acetonitrile/water=30/70at a flow rate of 0.5 ml/min, (2) an alkali eluent ofacetonitrile/aqueous 0.01 mol sodium hydroxide solution=50/50 is passedthrough the same column at a flow rate of 0.5 ml/min for 4 hours at acolumn temperature of 40° C., then an eluent of acetonitrile/aqueous0.1% phosphoric acid solution=30/70 is passed at a flow rate of 0.3ml/min for 30 minutes, the column temperature is set to 40° C., and thepyridine retentivity is measured by using an eluent ofacetonitrile/water=30/70 at a flow rate of 0.5 ml/min, and (3) thepyridine retentivity of (2) is compared with the pyridine retentivity of(1).
 18. The method for producing a modified polymer particle improvedin the acid-alkali durability as claimed in any one of claims 13 to 16,wherein the average particle size of the polymer particle is from 1 to50 μm.
 19. A method for producing a polymer-base packing material forchromatography, comprising producing the polymer-base packing materialfor chromatography described in claim 6 by performing one or both of thehydrolysis treatment with an acid or alkali hydrous solution and thecapping treatment through amidation, in the state of the polymer-basepacking material being packed in the column.
 20. A chromatography columnusing the polymer-base packing material for chromatography improved inthe acid-alkali durability described in claim 6.